The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is a medically important ABC drug transporter. Overexpression of BCRP in cancer cells is associated with resistance to multiple anticancer drugs, including mitoxantrone, topotecan, and flavopiridol. Correlation between BCRP expression and drug resistance or patient survival has been observed with some hematological and solid tumors. In addition, BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, and in the liver canalicular membrane. The tissue localization patterns, and results from recent studies, strongly suggest that BCRP functions as a protective efflux pump, and has the potential to limit oral absorption and increase biliary elimination of drugs that are BCRP substrates. At present, the molecular mechanism by which BCRP acts to transport drugs is unknown. Thus, the long-term goal of this proposal is to explore the molecular mechanism of BCRP. To achieve this goal, we propose one main specific aim, namely specific aim 1: structure and function analysis of BCRP. Site-directed mutagenesis and functional characterization of BCRP mutants, protein purification, and structure determination will be employed. The specific aim 1a is to purify BCRP overexpressed in the methylotrophic yeast Pichia pastoris. Purified protein will be functionally characterized and used for structural determination by electron microscopy. The specific aim 1b is mutational analyses to identify amino acid residues important for transport function and substrate selectivity of BCRP. The specific aim 1c is to analyze membrane topology of the transporter. Such studies will lead to a greater understanding of the molecular mechanism by which BCRP acts to transport drugs and provide the molecular basis for developing new ways to circumvent drug resistance in diseases such as cancers. Knowledge gained from the above studies will also aid in predicting potential drug-drug interactions and changes of pharmacokinetic properties of BCRP substrate drugs.